The long range goal of this project is to contribute to the understanding of the biochemical mechanisms through which the insulin receptor regulates cellular activities. Based on the preliminary results obtained by the principal investigator, the insulin receptor will be examined as a possible target of tyrosine phosphorylation by the src kinase under in vivo conditions. The objective is to study the impact of tyrosine phosphorylation on the bioactivity and dynamics of the insulin receptor independent of the binding of insulin. Various insulin receptor bearing and insulin responsive cell types will be selected on the basis of their ability to serve as hosts for the src kinase introduced by virus infection, plasmid DNA transfection or fusion with src kinase containing liposomes. The effects of the incorporated src kinase on the phosphorylation state of the insulin receptor will be investigated by tryptic phosphorylation mapping and phosphoamino acid analysis of phosphorylation sites. The phosphorylation characteristics of the insulin receptor in the src kinase containing cells will be compared to those of the receptor in control cell in response to insulin. Results to be obtained will indicate whether or not the src kinase will tyrosine phosphorylate the insulin receptor, and if so, whether the site(s) of src kinase-catalyzed phosphorylation are similar to those stimulated by insulin. The influence of tyrosine phosphorylation on insulin receptor serine and threonine influence of tyrosine phosphorylation on insulin receptor serine and threonine phosphorylation will also be studied to gain insight into the intramolecular regulation of receptor phosphorylation. To monitor any changes in cellular activities possibly associated with the src kinase-catalyzed tyrosine phosphorylation of the insulin receptor, basal and insulin-stimulated biological processes such as hexose and amino acid transport, IGF-II binding, glycogen synthase activity, insulin receptor cellular disposition and kinase activity and cell growth in src kinase containing cells will examined. Insulin receptor minus mutants will be generated and utilized as negative controls to test whether src kinase-catalyzed tyrosine phosphorylation of insulin receptor may be the primary cause of any changes in cellular metabolism. Finally, to assess directly the possible role of tyrosine phosphorylation in regulating the bioactivity and cellular disposition of the insulin receptor, differentially tyrosine phosphorylated receptor will be prepared under in vitro conditions, reconstituted into liposomes and fused into cells.
